No. 4535 September 29, 1956 NATURE 697 Pseudo-cholinesterase in Rat Erythrocytes basis of its substrate ratio, it is possible that it may be a butyro-cholinesterase. .ALTHOUGH there are many references in the literature to the existence of different types of cholin­ D.R. DAVIES esterase in mammalian sera1, it is generally believed J. P. RUTLAND 2 that human red cells contain only one type • Further­ more, we can find no evidence in the literature for Chemical Defence Experimental Establishment the existence of a pseudo-cholinesterase in any (Ministry of Supply), mammalian erythrocytes, although many workers Porton, Salisbury. have stated that benzoylcholine is hydrolysed by May 3. red cells. Since, however, the rate of hydrolysis is 1 Mundell, D. B., Nature, 153, 557 (1944). Ellis, S., Sanders, S., and slow, this breakdown of a substrate formerly presumed Bodansky, 0., J. Pharmacol., 91, 225 (1947). Levine M G and Suran, A. A., Enzymologia, 15, 17 (1951). ' · ., to be specific for pseudo-cholinesterase has been 'Mendell, B., Mundell, D. B., and Rudney, H., Biochem. J. 37 473 attributed to true cholinesterase. Indeed, Adams3 (1943). ' ' has shown that purified true cholinesterase does • Adams, D. H., Bwchim. Biophys. Acta, ~. 1 (1949). hydrolyse benzoylcholine, at a rate which would • Fraser, P. ~-, ti:,esis, University of Birmingham (1951), and private commumcat10n (1956). account for such observations. In spite of this, • Aldridge, W. N., Biochem. J., 53, 62 (1953). Austin, L., and Berry some results obtained by Fraser with benzoylcholine• W. K., Bwchem. J., 54, 695 (1953). ' imply the existence of more than one type of cholin­ 'Bayliss, B. J., and Todrick, A., Biochem. J., 62, 62 (1956). esterase in red cells. ' Sawyer, C. H., Science, 101, 385 (1945). Experiments which we have recently carried out 'Ord, M. G., and Thompson, R. H. S., Biochem. J., 49, 191 (1951). on erythrocytes twice washed with 0·9 per cent sodium chloride demonstrate quite clearly the existence of a true and pseudo-cholinesterase in rat red cells. The latter differ markedly from human Receptor Occupancy and Tissue Response erythrocytes in that they hydrolyse butyrylcholine ONE of the cornerstones of all quantitative as rapidly as acetylcholine (Table 1). applications of classical receptor theory has been the postulate that the response of a given organ or tissue Table 1. RELATIVE RATES OF HYDROLYSIS OF CHOLINE ESTERS BY RAT AND HUMAN RED CELLS is proportional to the number of receptors occupied Acetylcholine Acetyl-,8- Butyrylcholine Propionyl­ by the activating agent, and that the maximal methylcholine choline response occurs when all receptors are occupied. Rat 100 85 101 79 Human 100 78 10 63 Although never proved experimentally, and perhaps a priori somewhat unlikely in view of the complex This hydrolysis ofbutyrylcholine can be suppressed nature of tissue responses, this relationship is necess­ by tetraisopropylpyrophosphoramide at a concentra­ ary if agonist dose - response curves are to be ex­ tion (2 X 10-• M) which does not inhibit the hydro­ plained in terms of mass-action equilibria, and it lysis of acetyl-f,-methylcholine to any marked extent. provides the basis for differentiating reversible com­ Tetraisopropylpyrophosphoramide is an inhibitor petitive from non-competitive or irreversible in­ which has been shown6 to be specific for pseudo­ hibition. Basic formulations applying mass-action cholinesterase. 1 : 5 - bis - (4'trimethylammonium­ considerations to the differentiation of types of in­ phenyl)pentan-3-one diiodide (62047 Wellcome), a hibition are those of Gaddum1, applied largely to specific inhibitor of true cholinesterase6, at 7 x 10-• M tissue and organ responses, and of Linweaver and 2 inhibits the hydrolysis of acetyl-f,-methylcholine but Bllrke , which have been applied both to relatively not of butyrylcholine. simple enzyme systems and to complex responses of tissues and of intact animals. Table 2. INHIBITION OF HYDROLYSIS OF CHOLINE ESTERS BY RAT In general, use of the Linweaver-Burke equations RED CELLS IN THE PRESENCE OF INHIBITORS OF TRUE AND PSEUDO­ CHOLINESTERASE to evaluate data obtained from studies on enzyme Per cent inhibition ( calculated from the ratio systems has proved satisfactory. However, some of hydrolysis-rate In the presence, and absence, fundamental error in the application of these equa­ of inhibitor for the given substrate) tions to data obtained from experiments on .intact Inhibitor Acetyl- Acetyl-,8- Butyryl- Propionyl- choline methylcholine choline choline animals or isolated tissues is suggested by reports Tetraisopropylpyro­ which classify the f,-haloalkylamines (dibenamine phosphoramide 20 15 100 30 62047 92 97 0 45 congeners) as reversible competitive blocking agents 3 on this basis • Except at high concentrations of the We have insufficient evidence to enable us to draw inhibitor, data for the blockade of responses to any definite conclusions concerning the nature and adrenaline or histamine by these agents are typical source of the pseudo-enzyme. For example, we cannot of reversible competitive inhibition when plotted say whether it is similar to, or derived from, the by either of the methods mentioned above. This serum enzyme. It is certainly firmly attached to observation conflicts with several lines of evidence the cells, since it cannot be easily washed off. All the which indicate that the blockade, although it is experiments quoted were done on pooled erythrocytes 'competitive' in the sense that the agonist and the of male rats ; hence the presence of this enzyme in antagonist react with the same receptors, is predom­ female red cells has yet to be established. The precise inantly irreversible because of a stable bonding of the type of pseudo-cholinesterase, too, is a matter for inhibitor to some tissue constituent in the area of speculation. The almost complete lack of activity the receptor•. towards benzoylcholine makes it most unlikely that Two types of experiments have been combined it is a benzoylcholinesterase1, and the low propionyl­ in the present study to provide a possible explanation choline to acetylcholine ratio of O·8 would appear for the above discrepancy. Isolated strips of guinea to rule out the possibility of a propiono-cholinesterase pig ileum were exposed to GD-121 (SY-14, JlO; such as that found in rat heart•, and which has a N -l-naphthylmethyl-N -ethyl-f,-chloroethylamine) for corresponding value of approximately 2 ·3. On the 5 min., and the degree of blockade of the isotonic © 1956 Nature Publishing Group.